Interpretive Summary: Despite a recent new classification, a stable tree of life for the cycads has been elusive, particularly regarding relationships of Bowenia, Stangeria and Dioon. In this study we apply five single copy nuclear genes (SCNGs) to developing an accurate tree-of-life for the cycads, using several new statistical methodologies that reconcile individual gene trees with a consensus species tree. DNA sequences of five SCNGs were obtained for 20 cycad species representing all ten genera of cycads. We also estimated the ages of each genus using fossils as calibration points, and conducted a biogeographic analysis. All of our statistical methods converged on the same well-supported tree of life. However, massive extinction events inferred from the fossil record that eliminated broader distributions ranges within cycad lineages compromise accurate optimization of ancestral biogeographical areas. While major lineages of cycads are ancient, modern genera are no older than 12 million years. Our stable tree-of-life can be used to construct an accurate classification of the cycads.

Technical Abstract:
Despite a recent new classification, a stable tree of life for the cycads has been elusive, particularly regarding resolution of Bowenia, Stangeria and Dioon. In this study we apply five single copy nuclear genes (SCNGs) to the phylogeny of the order Cycadales. We specifically aim to evaluate several gene tree/species tree reconciliation approaches for developing an accurate phylogeny of the order, contrasting them with concatenated parsimony analysis, and resolve the erstwhile problematic phylogenetic position of these three genera. DNA sequences of five SCNGs were obtained for 20 cycad species representing all ten genera of Cycadales. These were analyzed with parsimony and three Bayesian methods of gene tree/species tree reconciliation, using Cycas as outgroup. A calibrated date estimation was developed with Bayesian methods, and biogeographic analysis was also conducted. Concatenated parsimony and three species tree inference methods resolve exactly the same tree topology with high support at most nodes. Dioon and Bowenia are the first and second branches of Cycadales after Cycas, respectively, followed by an encephalartoid clade (Macrozamia-Lepidozamia-Encephalartos), which is sister to a zamioid clade, of which Ceratozamia is the first branch, and in which Stangeria is sister to Microcycas and Zamia. We present a single, well supported phylogenetic hypothesis of the generic relationships of the Cycadales. However, massive extinction events inferred from the fossil record that eliminated broader distributions ranges within Zamiaceae compromise accurate optimization of ancestral biogeographical areas for that hypothesis. While major lineages of Cycadales are ancient, crown ages of all modern genera are no older than 12 million years, confirming a recent hypothesis of mostly Miocene radiations. Our stable phylogeny can inform an accurate infrafamilial classification of Zamiaceae.